Quadruplex DNA, or G4DNA, is made of four guanines that form Hoogsteen hydrogen bonds in a planar ring which is referred to as a G quartet. Multiple stacks of these G quartets (or tetrads) associate to form highly stable structures. Recently, several methodologies have provided convincing evidence confirming that G4DNA in cells affects DNA metabolism including transcription and replication. Recent work has provided a direct link between formation of G4DNA and G4RNA sequences and some neurodegenerative diseases such as ALS. Intense efforts are underway to develop agents that bind to G4DNA sequences for treatment of various cancers. G4DNA sequences are found throughout the genome, but are localized preferentially to certain regions such as promoters of proto-oncogenes, telomeres, and mitochondrial DNA. Despite the intense research focused on G4DNA, the mechanism(s) through which these structures impart biological function are largely unknown. We have applied a proteomic screen to discover new proteins that bind to G4DNA. The major proteins identified, including DHX36, are known to assemble into cytoplasmic structures termed stress granules under conditions of cellular stress. The location of these proteins and their known roles in regulation of translation leads us to a new hypothesis for the function of G4DNA. We propose that endogenous G4DNA excised from damaged mitochondrial and nuclear genomes can enter the cytosol intact. This excised G4DNA binds to proteins involved in translation to initiate a stress response through formation of stress granules. Our hypothesis provides a new mechanism by which cells can respond to the effects of DNA damage during oxidative stress. Hence, excised G4DNA can serve as a chemical signaling agent, alerting the cell to DNA damage. To test this hypothesis, we will determine the quantity and identity of G4DNA in the cytoplasm as a function of oxidative cell stress (Aim 1). In Aim 2, we will determine if G4DNA modulates the translation regulatory activity of DHX36 and if G4DNA and DHX36 co-localize with stress granule proteins. Aim 3 will focus on the consequences of different types of G4DNA on the enzymatic activity of DHX36 to test possible mechanisms by which G4DNA affects the protein. Different G4DNA structures will be evaluated in a biological screen that reports on translation. This work will have a direct impact on our understanding of the mechanisms of small molecules that are targeted to G4DNA and have been proposed as therapies for cancer treatment. Multiple research areas of relevance to cancer etiology will be impacted by this research including DNA damage response, mitochondrial and telomere biology, innate immunity, translation regulation, and cell signaling.